{"paper_id":"47079166-d733-4112-9d13-5256f082d8d9","body_text":"Behavioral, electrophysiological, and epigenetic characterization of acute cannabinoid treatment during pilocarpine-induced status epilepticus in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Behavioral, electrophysiological, and epigenetic characterization of acute cannabinoid treatment during pilocarpine-induced status epilepticus in mice Rafael Ignacio Gatica, Verónica Noches, Marcela Gonzalez, Pablo Henny, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9569465/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract There is growing interest in using cannabis and its active ingredients for epilepsy treatment. The main components of cannabis are cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), with some formulations of CBD approved for treating epilepsies refractory to traditional pharmacological treatments. However, it remains unclear whether the acute administration of THC or CBD can reverse a seizure once it has started. In this study, we used a temporal epilepsy model in mice induced by pilocarpine injection and analyzed the effects of CBD and THC during status epilepticus (SE) on behavior, cortical activity, and epigenetics. Behavioral analyses showed that CBD did not alter seizure severity, while THC only increased sedation. Regarding cortical electrical activity, neither CBD nor THC reverted the electroencephalographic changes induced by pilocarpine. Finally, we found that acute THC treatment prevented the seizure-induced homeostatic downregulation of neuroLSD1, maintaining it at physiological levels despite the insult. These results highlight that an acute dose of cannabinoid drugs is insufficient to reduce seizures and suggest that THC could promote the ongoing network excitability during seizures. epilepsy pilocarpine epigenetics neuroLSD1 THC CBD Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Epilepsy is a chronic neurological disorder affecting approximately seventy million people worldwide, with temporal lobe epilepsy (TLE) representing the most common and refractory focal syndrome in adults [ 1 , 2 ]. Despite the development of new anti-seizure medications, approximately one-third of patients develop drug-resistant epilepsy, defined as the failure to achieve sustained seizure freedom after two or more appropriate drug trials [ 3 , 4 ]. A critical manifestation of this condition is Status Epilepticus (SE), a state of unremitting seizure activity that becomes increasingly resistant to benzodiazepines the longer it persists [ 1 ]. The pilocarpine model in rodents serves as a high-isomorphism translational model for TLE, reproducing the complex temporal evolution of the human disease [ 5 ]. Systemic administration of this potent muscarinic agonist triggers intense limbic seizures via M1 receptor activation, as evidenced by studies in M1 receptor-deficient mice that are entirely resistant to the convulsant effects of the drug [ 6 ] This induction leads to a triphasic progression characterized by an acute period of SE, a latent (or silent) seizure-free period associated with circuit reorganization, and the eventual emergence of chronic, spontaneous recurrent seizures [ 5 ]. This model reproduces the acute pharmaco-resistant phase of SE characterized by pathological hypersynchrony in high-frequency beta (12–30 Hz) and gamma (30–70 Hz) EEG bands [ 7 ]. In the search for novel therapeutics, phytocannabinoids such as cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) have garnered significant attention, yet their efficacy appears highly context-dependent. Pharmaceutical-grade CBD has established significant success in treating genetic pediatric syndromes, consistently demonstrating statistically significant reductions in seizures for patients with Dravet and Lennox-Gastaut syndromes [ 8 , 9 ]. However, this success has not been translated to adult focal drug-resistant epilepsy, where recent randomized controlled trials found that transdermal CBD failed to outperform placebo [ 10 ]. This clinical divergence is also found in preclinical research. While CBD demonstrates robust efficacy in chemoconvulsant models like pentylenetetrazol [ 11 ] and genetic models of Dravet syndrome [ 12 ], its ability to halt the high-intensity glutamatergic drive of pilocarpine-induced SE appears limited. In models of structural pathology, the protective capacity of the endocannabinoid system is often overwhelmed [ 13 ], and acute CBD administration fails to terminate sustained epileptiform activity [ 14 ]. Furthermore, the utility of THC is complicated by a phenomenon called electro-clinical dissociation. In this condition, CB1 receptor agonists can induce a state of behavioral calm that conceals the visible symptoms of a seizure, even though the underlying hypersynchrony in the hippocampus continues to persist [ 15 , 16 ]. Consequently, relying solely on behavioral outcomes may obscure the persistence of network excitability and the molecular sequelae of SE. Beyond immediate seizure suppression, targeting epigenetic regulation offers a disease-modifying strategy [ 17 ]. Neuronal excitability is tightly governed by the epigenetic enzyme LSD1 (Lysine-Specific Demethylase 1/KDM1A) [ 18 ]. In the brain, a neurospecific splicing event involving the inclusion of microexon E8a generates neuroLSD1, a dominant-negative isoform essential for activity-dependent gene transcription and synaptic plasticity [ 19 , 20 ]. Pilocarpine-induced SE alters the ratio of LSD1 isoforms, potentially locking chromatin in a repressive state and forming a persistent epigenetic dysregulation [ 19 , 21 ]. This maladaptive splicing switch favors the repressive LSD1 complex, thereby silencing critical immediate early genes (IEGs) such as BDNF and c-Fos that are required for homeostatic plasticity and circuit remodeling [ 22 ]. Consequently, identifying pharmacological agents capable of restoring the physiological neuroLSD1/LSD1 ratio represents a novel avenue for neuroprotection, potentially mitigating the long-term comorbidities of epilepsy even in the absence of complete seizure control. While the anticonvulsant properties of cannabinoids are well-documented, their potential to modulate this specific epigenetic machinery during acute excitotoxicity remains unexplored. Therefore, this study aimed to characterize the behavioral, electrophysiological, and epigenetic effects of acute CBD and THC treatment in the pilocarpine model. We specifically investigated whether these cannabinoids could reverse the seizure-induced downregulation of the splicing variant neuroLSD1 and whether such molecular rescue correlates with the cessation of electrographic seizures. Material and methods Animals Male C57BL/6 mice, six weeks old (20–25 g), were obtained from the animal facility of the Faculty of Biological Sciences of the Pontificia Universidad Católica de Chile. All experimental procedures were approved by the Bioethical Committee of the Pontificia Universidad Católica de Chile (protocol ID 03082015). All procedures were carried out in strict accordance with the guidelines published in the “National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals” (8th edition) and the principles presented in the “Guidelines for the Use of Animals in Neuroscience Research” by the Society for Neuroscience. All procedures were conducted to minimize the number of animals used and their suffering. Animals were grouped in a climate-controlled vivarium on a 12-h light/dark cycle with food and water provided ad libitum . Pilocarpine model Animals were injected with the muscarinic receptor antagonist scopolamine methyl nitrate 1 mg/kg i.p (Sigma-Aldrich) to minimize peripheral pilocarpine-induced side effects. Pilocarpine 300 mg/kg i.p (Sigma-Aldrich) was injected 30 min (Fig. 1 ) or 15 min (Fig. 3 A) after scopolamine methyl nitrate. We selected this dose of pilocarpine because it induced seizures in all mice and did not require the administration of anticonvulsants. All animals were videotaped for 2 h to record their behavior, and the severity of seizure was evaluated using the Racine scale [ 23 ]. CBD/THC administration protocol Twenty minutes after the onset of pilocarpine-induced seizures, animals received a single injection of either CBD 100 mg/kg i.p or THC 10 mg/kg i.p (THC Pharm GmbH, Germany). The vehicle used consisted of Ethanol:Cremophor:NaCl 0.9% at a 1:1:18 ratio. Exon inclusion frequency by relative quantity Fluorescent-PCR analysis (Rqf-PCR) Total RNA was isolated from mice hippocampus 7 h after pilocarpine injection using Trizol reagent (Invitrogen Life Technologies) and reverse transcribed using MMulV (Thermo Scientific). Rqf-PCR was performed as previously described [ 24 ]. The ratio of neuroLSD1/LSD1 was analyzed by peak scanner software v.1.0. Electroencephalographic data acquisition and analysis Anesthesia was induced with isoflurane (Baxter Healthcare), followed by an injection of urethane 1.5 g/kg i.p (Sigma-Aldrich). Mice were then positioned in a stereotaxic apparatus adapted for mice using a MA-6N headholding adaptor (Narishige). Body temperature was maintained at 37°C with a homeothermic heating device (ATC 1000, World Precision Instruments). An incision at the base of the skull was treated with 20% topical benzocaine (Mayon) for pain relief and phosphate-buffered saline (PBS, pH 7.4) was applied prevent dehydration. A craniotomy was performed at the following coordinates: anteroposterior: -2.0 mm posterior to bregma, mediolateral: -1.5 mm lateral to midline. A stainless-steel screw was positioned as the recording electrode, and another screw was placed 1.5 mm posterior to lambda on the midline as the reference electrode. A ground electrode was placed under the exposed skin. EEG signals were amplified 2000X using a DPA-2FS (Scientifica), filtered (0.3–1500 Hz), and recorded in differential mode. Signals were digitized using a Power 1401 analog-to-digital converter (Cambridge Electronic Design) and Spike2 software (version 7.06, Cambridge Electronic Design) for data acquisition and online analysis, sampled at 1000 Hz. The recording protocol is shown in Fig. 3 A. Baseline recording lasted 5 min, followed by an injection of scopolamine methyl nitrate 1 mg/kg i.p (Sigma-Aldrich) with 15 min of activity recording. Then, pilocarpine 300 mg/kg i.p (Sigma-Aldrich) was injected, and after 20 min, vehicle, CBD 100 mg/kg i.p (THC Pharm GmbH, Germany), or THC 10 mg/kg i.p (THC Pharm GmbH, Germany) were administered. Recording lasted 2 h in total. Subsequently, anesthetized mice were euthanized by decapitation with a scissor. Data were extracted from Spike2 as MATLAB files and analyzed using MATLAB (version 2023a, MathWorks). Signals were downsampled to 500 Hz and the DC offset was removed. The 50 Hz noise was reduced using a notch filter. Spectral analysis was performed using previously published scripts [ 25 ] and the Chronux toolbox [ 26 ] with the following parameters: time-bandwidth product = 2, number of tapers = 3, frequency band = 0-150 Hz, moving window = 4 s (0.1 s overlap). Signal power was transformed to decibels. The following bandwidths were used for later analyses: delta (0.9-4.0 Hz), theta (4.0–8.0 Hz), alpha (8.0–12 Hz), beta (12–30 Hz), and gamma (30–70 Hz). The percentage of relative power was normalized against the 20 min preceding the pilocarpine injection and analyzed in 20- or 40- min bins, as required. No differences were found between the baseline and post-scopolamine injection periods (data not shown), therefore, these data were combined for analysis. Data analysis and figures Parametric or non-parametric ANOVA was used as appropriate. Statistical analyses were performed using GraphPad Prism (version 9, GraphPad Software). Figures were generated in GraphPad Prism and MATLAB. Results Effect of CBD and THC on seizures induced by pilocarpine Our study aimed to investigate the effects of the cannabinoid drugs CBD and THC on epilepsy in mice. Specifically, we induced temporal lobe epilepsy in mice using the muscarinic agonist pilocarpine [ 5 ]. We employed a protocol similar to the one described in our previous publication [ 21 ], as illustrated in Fig. 1 . To mitigate the peripheral effects of muscarinic agonism, we administered scopolamine methylnitrate 1 mg/kg i.p. Thirty minutes later, pilocarpine 300 mg/kg i.p was injected. Subsequently, after 20 min, mice received one of three treatments: vehicle, CBD 100 mg/kg i.p, or THC 10 mg/kg i.p. We video-recorded mouse behaviors during this period and measured two parameters: the duration of seizures and the Racine scale score. Seizure frequency was increased in the CBD group compared to vehicle and THC groups (Fig. 2 A, One-way ANOVA: F (2,19)= 8.924, p < 0.01; CBD vs vehicle or THC = p < 0.01 with Tukey's multiple comparisons test). However, we observed no changes between the vehicle and THC groups (Fig. 2 A). Regarding seizure duration parameters, THC groups showed decrease in total seizure duration (Fig. 2 B, One-way ANOVA: F (2,19)= 9.612, p < 0.01; vehicle vs THC = p < 0.01 with Tukey's multiple comparisons test) and mean seizure duration (Fig. 2 C, One-way ANOVA: F (2,19)= 5.836, p < 0.05; vehicle vs THC = p < 0.05 with Tukey's multiple comparisons test). Meanwhile, we found a decrease in the mean but not in total seizure duration for the CBD group (Fig. 2 B-C, mean seizure duration: vehicle vs CBD = p < 0.05 with Tukey's multiple comparisons test). Although these data show the effect of CBD in pilocarpine-induced seizures, evaluation of seizure severity with the Racine scale showed no effect of CBD on seizure (Fig. 2 D). Meanwhile, a decrease in Racine scale value was found in THC group (Fig. 2 D, Kruskal-Wallis test: p < 0.01; vehicle and CBD vs THC = p < 0.05 with Dunn's multiple comparisons test), but it is relevant to note that animals exhibited marked sedation after THC injection. Overall, these behavioral data indicate that CBD decreases the duration but not the severity of seizures, while THC decreases seizure severity and duration, increasing mice drowsiness. CBD and THC do not alter pilocarpine-induced cortical activity We assessed the effects of CBD and THC on cortical activity using EEG in anesthetized mice. Figure 3 A summarizes the experimental protocol. Under urethane anesthesia, a screw electrode was placed at the cortical level, near the dorsal hippocampus (for more details, see the Methods section). To minimize the peripheral effects of pilocarpine, mice were injected with scopolamine methylnitrate. Subsequently, pilocarpine 300 mg/kg i.p was administered, and 20 min later, either vehicle, CBD (100 mg/kg, i.p), or THC (10 mg/kg, i.p) was injected. Recordings were conducted for 2 h. A representative example of an EEG recording 20 min before and after pilocarpine 300 mg/kg i.p. injection is shown in Fig. 3 B, C. During baseline, the raw signal showed high-amplitude low frequency oscillations, which are characteristic of urethane anesthesia [ 27 ] ( Fig. 3B1 ). Following pilocarpine injection, high frequency oscillations were more prominent ( Fig. 3B2 ). Spectral analysis confirmed these observations, as frequencies lower than 5 Hz showed high power values during baseline ( Fig. 3C1 ), whereas the power of frequencies higher than 10 Hz were increased after pilocarpine ( Fig. 3C2 ). To evaluate the effect of pilocarpine in specific bandwidths, we performed statistical analyses, considering all groups together (Fig. 3 D, N = 13). We found a significant increase in the percentage of relative power was found at theta (4–8 Hz), beta (12–30 Hz) and gamma (30–70 Hz) frequencies (Fig. 3 D, p < 0.001 with Wilcoxon sign rank test). We found comparable results when raw EEG power was analyzed or when this analysis was performed between groups (data not shown). Overall, these data show that a pilocarpine injection under anesthesia can induce epileptic-like activity in the EEG. We studied the effect of CBD and THC on EEG activity after pilocarpine injection (Fig. 4 ). The median EEG spectral power was obtained and color-plotted over time for vehicle (N = 5), CBD 100 mg/kg i.p (N = 4) and THC 10 mg/kg i.p (N = 4) groups (Fig. 4 A). Before and after treatment administration (at 40 min), we observed no differences between groups in the spectral density maps (Fig. 4 A). Then, we calculated the percentage of relative power for all bandwidths, in the 40–80 (Fig. 4 B1 ) and 80–120 ( Fig. 5B2 ) minute epochs. All frequency bands (except alpha) exhibited an increase in power compared to the baseline (pre-pilocarpine). The most significant increases were observed in the beta (up to 500%) and gamma (up to 1000%) bands. Between groups, neither CBD nor THC induced changes in EEG power bands compared to the vehicle, finding no statistically significant differences in any band for each bin tested (Fig. 4 B1-2, Kruskal-Wallis test: p > 0.05). These findings indicate that while pilocarpine significantly elevates the power across all EEG frequency bands, particularly in the high-frequency beta and gamma ranges, neither CBD nor THC alters this heightened cortical activity. This shows that under the conditions of our study, CBD and THC do not modulate the acute electroencephalographic responses induced by pilocarpine. THC, but not CBD, reverses pilocarpine-induced changes in neuroLSD1 levels We have previously demonstrated that pilocarpine leads to significant epigenetic modifications in the hippocampus, including alterations in the LSD1/CoREST/HDAC1/2 complex and histone methylation patterns [ 21 ]. In this study, we further investigated changes in the neuroLSD1/LSD1 ratio. Seven hours after pilocarpine injection, mice were sacrificed, and the prefrontal cortex (PFC) and hippocampus were dissected to extract mRNA (Fig. 1 , for more details in Methods). We measured the percentages of neuroLSD1 and LSD1 for each group (Fig. 5 ). A vehicle group was included for comparison. One-way ANOVA showed significant differences in both the hippocampus (F (3, 12) = 6.881, p = 0.0060) and PFC (F (3, 12) = 5.787, p = 0.0110). We observed a decrease in neuroLSD1 transcript after pilocarpine injection compared to control conditions in the hippocampus (Fig. 5 A, p < 0.05 Tukey's multiple comparisons test) but not in the PFC (Fig. 5 B, p > 0.05 Tukey's multiple comparisons test). These hippocampal results replicate our previous findings [ 21 ]. Regarding cannabinoid drugs, CBD did not significantly alter LSD1 levels compared to vehicle or pilocarpine alone in either region (Fig. 5 , p > 0.05, Tukey's multiple comparisons test). Notably, THC increased neuroLSD1 levels compared to both pilocarpine and pilocarpine + CBD in the hippocampus and PFC (Fig. 5 , p < 0.05, Tukey's multiple comparisons test). In summary, while CBD did not affect LSD1 levels, THC significantly increased neuroLSD1 expression in both regions, suggesting a potential differential impact of cannabinoids on the epigenetic landscape following seizure induction. DISCUSSION The present study reveals critical dissociation among behavioral, electrophysiological, and genomic effects of acute cannabinoid administration during a SE induced by pilocarpine. Our main findings indicate that neither CBD nor THC was sufficient to normalize cortical electroencephalographic activity during SE, although both drugs reduced the duration of seizures. Furthermore, THC inhibited the negative homeostatic regulation of neuroLSD1, suggesting the maintenance of cortical excitability, which may facilitate further seizures. Regarding behavioral effects, the cannabinoids elicited distinct phenotypes. THC induced a marked sedation [ 28 ] characteristic of CB1 receptor activation [ 29 , 30 ], which effectively masked the behavioral expression of seizures. In contrast, CBD (100 mg/kg) failed to reduce seizure severity, diverging from previous reports demonstrating efficacy at lower doses [ 14 ]. Instead, CBD produced a pattern of \"seizure fragmentation,\" characterized by a paradoxical increase in seizure frequency coupled with reduced individual seizure duration. This likely reflects the inverted U-shaped dose-response curve of cannabinoids [ 31 ]. We propose that while CBD may activate termination mechanisms via targets like ENT1, the concurrent engagement of pro-excitatory TRPV1 channels at high concentrations [ 32 ] lowers the seizure threshold, converting continuous status epilepticus into repetitive, fragmented events rather than achieving sustained seizure freedom. From an electrophysiological standpoint, the resistance to treatment was significant (Fig. 4 ). Despite the behavioral suppression seen with THC, neither cannabinoid reversed the pathological increase in beta (12–30 Hz) and gamma (30–70 Hz) EEG power. This persistence of the sustained epileptiform activity aligns with the hypothesis of homeostatic downregulation of the endocannabinoid system during severe excitotoxicity. Intense neuronal firing triggers the rapid internalization and desensitization of presynaptic CB1 receptors, rendering the system refractory to acute agonist stimulation precisely at the seizure focus [ 33 , 34 ]. It is important to acknowledge that these recordings were obtained under urethane anesthesia. Urethane potentiates GABA-A receptors and inhibits NMDA receptors, shifting the basal balance toward inhibition [ 35 ]. Although this could theoretically lead to a \"floor effect\" [ 36 ], our data indicate that pilocarpine caused substantial and sustained increases in high-frequency power that easily surpassed anesthetic suppression (Fig. 3 ). This phenomenon has been corroborated in other chemoconvulsant models, such as 4-aminopyridine, where high-frequency ictal discharges displace anesthetic slow-waves [ 27 ]. Furthermore, seminal work by Stringer & Sowell, 1994 confirms that urethane anesthesia preserves the validity of electrographic seizure patterns compared to awake animals, reinforcing that the failure of cannabinoids to reduce EEG power here was due to the refractory nature of the seizure network rather than an artifact of the anesthetic regimen. The disparity between the sedative effects of THC (Fig. 2 ) and its failure to reduce EEG power (Fig. 4 ) highlights a significant risk in preclinical drug evaluation. This uncoupling has been described with synthetic cannabinoids, where motor seizures are suppressed while hippocampal paroxysms persist [ 15 , 16 ]. Our results extend this to phytocannabinoids, serving as a critical methodological warning: behavioral quiescence induced by CB1 agonists should not be interpreted as seizure cessation without corroborating electrophysiological evidence. Despite the lack of electrographic silence, a key molecular finding was that THC, but not CBD, prevented the seizure-induced downregulation of neuroLSD1 (Fig. 5 ). NeuroLSD1 is a dominant-negative splicing variant of the epigenetic eraser LSD1 that facilitates the transcription of neuroplasticity genes [ 19 , 20 ]. The downregulation of neuroLSD1 observed in our vehicle group represents a maladaptive splicing switch that contributes to a global repressive state [ 21 ]. However, this molecular rescue presents a biological paradox when interpreted in the context of genetic studies. NeuroLSD1-null mice exhibit a hypoexcitable phenotype and increased resistance to pilocarpine-induced seizures [ 19 ]. This suggests that the drastic downregulation of neuroLSD1 we observed in the vehicle group may act as an adaptive homeostatic brake: an attempt by the network to limit excitability by reinstating the repressive LSD1 complex. Then, the effect of THC is double-edged. By restoring neuroLSD1 levels, THC effectively opposed this homeostatic downregulation. The specific ability of THC, but not CBD, to restore neuroLSD1 levels implies a mechanism whereby CB1 signaling exerts upstream control over the splicing machinery. While previous studies have shown that LSD1 regulates the endocannabinoid system [ 38 ], our data suggests a bidirectional relationship, potentially mediated by the CB1-dependent activation of MAPK/ERK pathways [ 39 ], which are known to regulate splicing factors. Conversely, the failure of CBD to rescue neuroLSD1 is consistent with its non-canonical pharmacological profile. Lacking intrinsic affinity for the CB1 receptor, CBD acts via a pleiotropic mechanism, involving targets such as GPR55, TRPV1, and ENT1 [ 40 ], which likely bypasses the specific CB1-dependent MAPK/ERK signaling axis required to drive the splicing machinery. Ultimately, while the restoration of neuroLSD1 by THC may preserve the chromatin competence required for immediate early gene transcription (potentially mitigating the \"epigenetic scar\"), it may simultaneously maintain the permissive state for network hypersynchrony, offering a molecular explanation for the observed electro-clinical dissociation. Declarations Funding This study was funded by: ANID Fondecyt Postdoctorado 3160308 to V.N. and 3230573 to R.I.G, ANID Fondecyt Regular 1191497 to P.H. Competing Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Author contributions Rafael Ignacio Gatica: conceptualization, methodology, writing – original draft, data acquisition, formal analysis, and investigation. Verónica Noches: conceptualization, methodology, writing-review & editing, data acquisition, formal analysis, investigation, funding acquisition. Marcela González: investigation, project administration. Pablo Henny: funding acquisition, supervision, writing-review & editing. María Estela Andrés: conceptualization, methodology, supervision, writing-review & editing, resources, funding acquisition. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval All experimental procedures were approved by the Bioethical Committee of the Pontificia Universidad Católica de Chile (protocol ID 03082015). 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Elife 4:e06513. https://doi.org/10.7554/eLife.06513 Bokil H, Andrews P, Kulkarni JE, Mehta S, Mitra PP, Chronux (2010) A platform for analyzing neural signals. J Neurosci Methods 192:146–151. https://doi.org/10.1016/j.jneumeth.2010.06.020 Tukacs V, Mittli D, Hunyadi-Gulyás É, Darula Z, Juhász G, Kardos J et al Comparative analysis of hippocampal extracellular space uncovers widely altered peptidome upon epileptic seizure in urethane-anaesthetized rats. Fluids Barriers CNS 2024;21. https://doi.org/10.1186/s12987-024-00508-w Báez-Cordero AS, Pimentel-Farfan AK, Peña-Rangel T, Rueda-Orozco PE (2020) Unbalanced Inhibitory/Excitatory Responses in the Substantia Nigra Pars Reticulata Underlie Cannabinoid-Related Slowness of Movements. J Neurosci 40:5769. https://doi.org/10.1523/JNEUROSCI.0045-20.2020 Herkenham M, Lynn AB, Little MD, Johnson MR, Melvin LS, de Costa BR et al (1990) Cannabinoid receptor localization in brain. Proceedings of the National Academy of Sciences. ;87:1932–6. https://doi.org/10.1073/pnas.87.5.1932 Tsou K, Brown S, Sañudo-Peña MC, Mackie K, Walker JM (1998) Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience 83:393–411. https://doi.org/https://doi.org/10.1016/S0306-4522(97)00436-3 Zavala-Tecuapetla C, Luna-Munguia H, López-Meraz M-L, Cuellar-Herrera M (2022) Advances and challenges of cannabidiol as an anti-seizure strategy: preclinical evidence. Int J Mol Sci 23:16181. https://doi.org/10.3390/ijms232416181 Vilela LR, Lima IV, Kunsch ÉB, Pinto HPP, de Miranda AS, Vieira ÉLM et al (2017) Anticonvulsant effect of cannabidiol in the pentylenetetrazole model: Pharmacological mechanisms, electroencephalographic profile, and brain cytokine levels. Epilepsy Behav 75:29–35. https://doi.org/https://doi.org/10.1016/j.yebeh.2017.07.014 Colangeli R, Morena M, Werner A, Thompson RJ, van der Stelt M, Pittman QJ et al (2023) 2-AG-Mediated Control of GABAergic Signaling Is Impaired in a Model of Epilepsy. J Neurosci 43:571–583. https://doi.org/10.1523/JNEUROSCI.0541-22.2022 Sugaya Y, Kano M (2022) Endocannabinoid-Mediated Control of Neural Circuit Excitability and Epileptic Seizures. Front Neural Circuits 15:781113. https://doi.org/10.3389/fncir.2021.781113 Hara K, Harris RA (2002) The Anesthetic Mechanism of Urethane: The Effects on Neurotransmitter-Gated Ion Channels. Anesth Analg 94. https://doi.org/10.1213/00000539-200202000-00015 Rojas A, Wang W, Glover A, Manji Z, Fu Y, Dingledine R Beneficial outcome of urethane treatment following status epilepticus in a rat organophosphorus toxicity model. ENeuro 2018;5. https://doi.org/10.1523/ENEURO.0070-18.2018 Stringer JL, Sowell KL (1994) Kainic acid, bicuculline, pentylenetetrazol and pilocarpine elicit maximal dentate activation in the anesthetized rat. Epilepsy Res 18:11–21. https://doi.org/10.1016/0920-1211(94)90029-9 Longaretti A, Forastieri C, Gabaglio M, Rubino T, Battaglioli E, Rusconi F (2020) Termination of acute stress response by the endocannabinoid system is regulated through lysine-specific demethylase 1-mediated transcriptional repression of 2-AG hydrolases ABHD6 and MAGL. J Neurochem 155:98–110. https://doi.org/10.1111/jnc.15000 Derkinderen P, Valjent E, Toutant M, Corvol J-C, Enslen H, Ledent C et al (2003) Regulation of Extracellular Signal-Regulated Kinase by Cannabinoids in Hippocampus. J Neurosci 23:2371. https://doi.org/10.1523/JNEUROSCI.23-06-02371.2003 Gray RA, Whalley BJ (2020) The proposed mechanisms of action of CBD in epilepsy. Epileptic Disord 22:S10–S15. https://doi.org/10.1684/epd.2020.1135 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-9569465\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":635788500,\"identity\":\"a872335c-76d5-4368-a24c-effd17f6aaa9\",\"order_by\":0,\"name\":\"Rafael Ignacio Gatica\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Arturo Prat University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Rafael\",\"middleName\":\"Ignacio\",\"lastName\":\"Gatica\",\"suffix\":\"\"},{\"id\":635788501,\"identity\":\"7a166ea6-5727-4a3e-94d0-46307f13938b\",\"order_by\":1,\"name\":\"Verónica Noches\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Western University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Verónica\",\"middleName\":\"\",\"lastName\":\"Noches\",\"suffix\":\"\"},{\"id\":635788502,\"identity\":\"5e72dbc8-2f27-4af0-9933-929783ab17ed\",\"order_by\":2,\"name\":\"Marcela Gonzalez\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Pontificia Universidad Católica de Chile\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Marcela\",\"middleName\":\"\",\"lastName\":\"Gonzalez\",\"suffix\":\"\"},{\"id\":635788503,\"identity\":\"cd95a4d4-3d68-487d-a898-ba28cdf9e102\",\"order_by\":3,\"name\":\"Pablo Henny\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Chile\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Pablo\",\"middleName\":\"\",\"lastName\":\"Henny\",\"suffix\":\"\"},{\"id\":635788504,\"identity\":\"bee44ea8-18ef-4b2a-8254-c11649b9f902\",\"order_by\":4,\"name\":\"María Estela Andrés\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAmElEQVRIiWNgGAWjYJACCSCWA7MekKLFGMxKIEVLYgPRWnTbzx68XVFRl77h+AHGD0RpMTuTl2x55szh3A1nEpgliNNyIMdMsrHtQO6GGwxsxDnM7PwboJZ/dekGxGu5AbKlgTmBFC1vjC0bjh02nHkmsZlIv5zPMbzZUFMnz3f88MEPH4jRggQYG0jUMApGwSgYBaMAJwAA060zbyXkqf8AAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Pontificia Universidad Católica de Chile\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"María\",\"middleName\":\"Estela\",\"lastName\":\"Andrés\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-04-29 19:38:29\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-9569465/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-9569465/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":108977579,\"identity\":\"665466f6-9488-48fa-9c34-ef1da207039a\",\"added_by\":\"auto\",\"created_at\":\"2026-05-11 11:32:11\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":940465,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eExperimental procedure.\\u003c/strong\\u003e Mice were injected with scopolamine methylnitrate 1 mg/kg i.p, followed by pilocarpine 300 mg/kg i.p 30 min after. Twenty minutes after pilocarpine injection, vehicle, CBD 100 mg/kg i.p or THC 10 mg/kg i.p were injected. Mice were video-recorded for 2 h in total and behavior was subsequently analyzed. Brain samples for mRNA analysis were obtained 7 h after pilocarpine injection.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig1Methodsv1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9569465/v1/e6e0e779f32b85e8f974fe5e.png\"},{\"id\":108832601,\"identity\":\"8aeacbc0-cebc-4cda-b072-b16ab743d0b3\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 20:34:12\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":6342105,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEffect of CBD and THC in behavioral seizures.\\u003c/strong\\u003e A) Seizure frequency during 2 h after pilocarpine 300 mg/kg i.p injection plus vehicle, CBD 100 mg/kg i.p or THC 10 mg/kg i.p. ** p\\u0026lt;0.01 one way ANOVA with Bonferroni’s multiple comparisons test. B) Total time in seizure. ** p\\u0026lt;0.01 one way ANOVA with Bonferroni’s multiple comparisons test. C) Duration of seizure. * p\\u0026lt;0.05 one way ANOVA with Bonferroni’s multiple comparisons test. Data are represented as bars showing mean ± SEM and individual values. D) Racine scale. * p\\u0026lt;0.05 Kruskal-Wallis test with Dunn’s multiple comparisons test. Data are represented as bars showing median, interquartile range and individual values.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig2Behaviorv2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9569465/v1/d04e065fbf9e21b8ab327b42.png\"},{\"id\":108832605,\"identity\":\"11105476-3f98-4450-a142-84d2e5f893f8\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 20:34:13\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":22989786,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEEG activity after pilocarpine 300 mg/kg i.p injection. \\u003c/strong\\u003eA) Experimental protocol. After anesthesia induction using urethane 1.5 g/kg i.p, baseline activity was recorded for 5 min. Next, scopolamine methylnitrate 1 mg/kg i.p was injected. Fifteen minutes later, pilocarpine 300 mg/kg i.p was injected. After 20 min, vehicle, CBD 100 mg/kg i.p or THC 10 mg/kg i.p were injected and EEG data was recorded until 120 min. B) Representative raw EEG signal during baseline (B1) and after pilocarpine 300 mg/kg i.p (B2). C) Representative spectrograms during baseline (C1) and after pilocarpine 300 mg/kg i.p (C2). Each color plot is normalized in decibel scale. D) Percentage of relative power (vs Baseline) of all mice during the first 20 min after pilocarpine 300 mg/kg i.p. *** p\\u0026lt;0.001 vs baseline of each band, using a Wilcoxon sign rank test. Data are represented as bars showing median, interquartile range and individual values.We studied the effect of CBD and THC on EEG activity after pilocarpine injection (\\u003cstrong\\u003eFig. 4\\u003c/strong\\u003e). The median EEG spectral power was obtained and color-plotted over time for vehicle (N=5), CBD 100 mg/kg i.p (N=4) and THC 10 mg/kg i.p (N=4) groups (\\u003cstrong\\u003eFig. 4A\\u003c/strong\\u003e). Before and after treatment administration (at 40 min), we observed no differences between groups in the spectral density maps (\\u003cstrong\\u003eFig. 4A\\u003c/strong\\u003e). Then, we calculated the percentage of relative power for all bandwidths, in the 40-80 (\\u003cstrong\\u003eFig. 4B1\\u003c/strong\\u003e) and 80-120 (\\u003cstrong\\u003eFig. 5B2\\u003c/strong\\u003e) minute epochs. All frequency bands (except alpha) exhibited an increase in power compared to the baseline (pre-pilocarpine). The most significant increases were observed in the beta (up to 500%) and gamma (up to 1000%) bands. Between groups, neither CBD nor THC induced changes in EEG power bands compared to the vehicle, finding no statistically significant differences in any band for each bin tested (\\u003cstrong\\u003eFig. 4B1-2\\u003c/strong\\u003e, Kruskal-Wallis test: p\\u0026gt;0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig3EEGBasalvsPilo.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9569465/v1/faf63913066c52b9a1d59ac1.png\"},{\"id\":108832603,\"identity\":\"c7f32a3b-7b75-4884-8db0-3c9e2bb068e5\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 20:34:12\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":46784904,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTHC and CBD did not modify EEG signals induced by pilocarpine. \\u003c/strong\\u003eA) Spectrograms showing the median power of vehicle (A1), CBD 100 mg/kg (A2) and THC 10 mg/kg (A3) treated mice. Each color plot is normalized in decibel scale.B) Percentage of relative power (to baseline) was analyzed in five bandwidths (Delta: 0.9-4.0 Hz; Theta: 4-8 Hz; Alpha: 8-12 Hz; Beta: 12-30 Hz; Gamma: 30-70 Hz) in 40 min windows: B1) 40-80 mi; B2) 60-80 min. Data are represented as bars showing median, interquartile range and Individual values.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig4EEGTratamientos.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9569465/v1/02d07207a9d93cf7a0a26b48.png\"},{\"id\":108832604,\"identity\":\"2ab696f1-45a8-42c7-b264-18d3cc6d1228\",\"added_by\":\"auto\",\"created_at\":\"2026-05-08 20:34:12\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":7717914,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTHC reverses pilocarpine induced changes in nLSD1.\\u003c/strong\\u003e The proportion of the neuronal variant (nLSD1) and ubiquitous LSD1 (uLSD1) transcripts assessed by Rqf-PCR after 7 h of vehicle, pilocarpine, pilocarpine plus CBD 100 mg/kg i.p or pilocarpine plus THC 10 mg/kg i.p in hippocampus (A) or PFC (B). * p ˂ 0.005 one-way ANOVA with Tukey’s multiple comparisons test. Data are represented as stacked bars showing mean ± SEM.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig5nLSD1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9569465/v1/f994501628ae7619b67e41aa.png\"},{\"id\":109081525,\"identity\":\"e5e9c32c-b3ab-4588-9a4d-c268fd42f436\",\"added_by\":\"auto\",\"created_at\":\"2026-05-12 12:19:46\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":78664391,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9569465/v1/3299dbfe-6a6d-4f08-b74d-7c3131b82678.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Behavioral, electrophysiological, and epigenetic characterization of acute cannabinoid treatment during pilocarpine-induced status epilepticus in mice\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eEpilepsy is a chronic neurological disorder affecting approximately seventy million people worldwide, with temporal lobe epilepsy (TLE) representing the most common and refractory focal syndrome in adults [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Despite the development of new anti-seizure medications, approximately one-third of patients develop drug-resistant epilepsy, defined as the failure to achieve sustained seizure freedom after two or more appropriate drug trials [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. A critical manifestation of this condition is Status Epilepticus (SE), a state of unremitting seizure activity that becomes increasingly resistant to benzodiazepines the longer it persists [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. The pilocarpine model in rodents serves as a high-isomorphism translational model for TLE, reproducing the complex temporal evolution of the human disease [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Systemic administration of this potent muscarinic agonist triggers intense limbic seizures via M1 receptor activation, as evidenced by studies in M1 receptor-deficient mice that are entirely resistant to the convulsant effects of the drug [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e] This induction leads to a triphasic progression characterized by an acute period of SE, a latent (or silent) seizure-free period associated with circuit reorganization, and the eventual emergence of chronic, spontaneous recurrent seizures [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. This model reproduces the acute pharmaco-resistant phase of SE characterized by pathological hypersynchrony in high-frequency beta (12\\u0026ndash;30 Hz) and gamma (30\\u0026ndash;70 Hz) EEG bands [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn the search for novel therapeutics, phytocannabinoids such as cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) have garnered significant attention, yet their efficacy appears highly context-dependent. Pharmaceutical-grade CBD has established significant success in treating genetic pediatric syndromes, consistently demonstrating statistically significant reductions in seizures for patients with Dravet and Lennox-Gastaut syndromes [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. However, this success has not been translated to adult focal drug-resistant epilepsy, where recent randomized controlled trials found that transdermal CBD failed to outperform placebo [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. This clinical divergence is also found in preclinical research. While CBD demonstrates robust efficacy in chemoconvulsant models like pentylenetetrazol [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e] and genetic models of Dravet syndrome [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e], its ability to halt the high-intensity glutamatergic drive of pilocarpine-induced SE appears limited. In models of structural pathology, the protective capacity of the endocannabinoid system is often overwhelmed [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e], and acute CBD administration fails to terminate sustained epileptiform activity [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. Furthermore, the utility of THC is complicated by a phenomenon called electro-clinical dissociation. In this condition, CB1 receptor agonists can induce a state of behavioral calm that conceals the visible symptoms of a seizure, even though the underlying hypersynchrony in the hippocampus continues to persist [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. Consequently, relying solely on behavioral outcomes may obscure the persistence of network excitability and the molecular sequelae of SE.\\u003c/p\\u003e \\u003cp\\u003eBeyond immediate seizure suppression, targeting epigenetic regulation offers a disease-modifying strategy [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Neuronal excitability is tightly governed by the epigenetic enzyme LSD1 (Lysine-Specific Demethylase 1/KDM1A) [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. In the brain, a neurospecific splicing event involving the inclusion of microexon E8a generates neuroLSD1, a dominant-negative isoform essential for activity-dependent gene transcription and synaptic plasticity [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Pilocarpine-induced SE alters the ratio of LSD1 isoforms, potentially locking chromatin in a repressive state and forming a persistent epigenetic dysregulation [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. This maladaptive splicing switch favors the repressive LSD1 complex, thereby silencing critical immediate early genes (IEGs) such as BDNF and c-Fos that are required for homeostatic plasticity and circuit remodeling [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Consequently, identifying pharmacological agents capable of restoring the physiological neuroLSD1/LSD1 ratio represents a novel avenue for neuroprotection, potentially mitigating the long-term comorbidities of epilepsy even in the absence of complete seizure control.\\u003c/p\\u003e \\u003cp\\u003eWhile the anticonvulsant properties of cannabinoids are well-documented, their potential to modulate this specific epigenetic machinery during acute excitotoxicity remains unexplored. Therefore, this study aimed to characterize the behavioral, electrophysiological, and epigenetic effects of acute CBD and THC treatment in the pilocarpine model. We specifically investigated whether these cannabinoids could reverse the seizure-induced downregulation of the splicing variant neuroLSD1 and whether such molecular rescue correlates with the cessation of electrographic seizures.\\u003c/p\\u003e\"},{\"header\":\"Material and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAnimals\\u003c/h2\\u003e \\u003cp\\u003eMale C57BL/6 mice, six weeks old (20\\u0026ndash;25 g), were obtained from the animal facility of the Faculty of Biological Sciences of the Pontificia Universidad Cat\\u0026oacute;lica de Chile. All experimental procedures were approved by the Bioethical Committee of the Pontificia Universidad Cat\\u0026oacute;lica de Chile (protocol ID 03082015). All procedures were carried out in strict accordance with the guidelines published in the \\u0026ldquo;National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals\\u0026rdquo; (8th edition) and the principles presented in the \\u0026ldquo;Guidelines for the Use of Animals in Neuroscience Research\\u0026rdquo; by the Society for Neuroscience. All procedures were conducted to minimize the number of animals used and their suffering. Animals were grouped in a climate-controlled vivarium on a 12-h light/dark cycle with food and water provided \\u003cem\\u003ead libitum\\u003c/em\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003ePilocarpine model\\u003c/h3\\u003e\\n\\u003cp\\u003eAnimals were injected with the muscarinic receptor antagonist scopolamine methyl nitrate 1 mg/kg i.p (Sigma-Aldrich) to minimize peripheral pilocarpine-induced side effects. Pilocarpine 300 mg/kg i.p (Sigma-Aldrich) was injected 30 min (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e) or 15 min (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA) after scopolamine methyl nitrate. We selected this dose of pilocarpine because it induced seizures in all mice and did not require the administration of anticonvulsants. All animals were videotaped for 2 h to record their behavior, and the severity of seizure was evaluated using the Racine scale [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e].\\u003c/p\\u003e\\n\\u003ch3\\u003eCBD/THC administration protocol\\u003c/h3\\u003e\\n\\u003cp\\u003eTwenty minutes after the onset of pilocarpine-induced seizures, animals received a single injection of either CBD 100 mg/kg i.p or THC 10 mg/kg i.p (THC Pharm GmbH, Germany). The vehicle used consisted of Ethanol:Cremophor:NaCl 0.9% at a 1:1:18 ratio.\\u003c/p\\u003e\\n\\u003ch3\\u003eExon inclusion frequency by relative quantity Fluorescent-PCR analysis (Rqf-PCR)\\u003c/h3\\u003e\\n\\u003cp\\u003eTotal RNA was isolated from mice hippocampus 7 h after pilocarpine injection using Trizol reagent (Invitrogen Life Technologies) and reverse transcribed using MMulV (Thermo Scientific). Rqf-PCR was performed as previously described [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. The ratio of neuroLSD1/LSD1 was analyzed by peak scanner software v.1.0.\\u003c/p\\u003e\\n\\u003ch3\\u003eElectroencephalographic data acquisition and analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eAnesthesia was induced with isoflurane (Baxter Healthcare), followed by an injection of urethane 1.5 g/kg i.p (Sigma-Aldrich). Mice were then positioned in a stereotaxic apparatus adapted for mice using a MA-6N headholding adaptor (Narishige). Body temperature was maintained at 37\\u0026deg;C with a homeothermic heating device (ATC 1000, World Precision Instruments). An incision at the base of the skull was treated with 20% topical benzocaine (Mayon) for pain relief and phosphate-buffered saline (PBS, pH 7.4) was applied prevent dehydration. A craniotomy was performed at the following coordinates: anteroposterior: -2.0 mm posterior to bregma, mediolateral: -1.5 mm lateral to midline. A stainless-steel screw was positioned as the recording electrode, and another screw was placed 1.5 mm posterior to lambda on the midline as the reference electrode. A ground electrode was placed under the exposed skin. EEG signals were amplified 2000X using a DPA-2FS (Scientifica), filtered (0.3\\u0026ndash;1500 Hz), and recorded in differential mode. Signals were digitized using a Power 1401 analog-to-digital converter (Cambridge Electronic Design) and Spike2 software (version 7.06, Cambridge Electronic Design) for data acquisition and online analysis, sampled at 1000 Hz. The recording protocol is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA. Baseline recording lasted 5 min, followed by an injection of scopolamine methyl nitrate 1 mg/kg i.p (Sigma-Aldrich) with 15 min of activity recording. Then, pilocarpine 300 mg/kg i.p (Sigma-Aldrich) was injected, and after 20 min, vehicle, CBD 100 mg/kg i.p (THC Pharm GmbH, Germany), or THC 10 mg/kg i.p (THC Pharm GmbH, Germany) were administered. Recording lasted 2 h in total. Subsequently, anesthetized mice were euthanized by decapitation with a scissor.\\u003c/p\\u003e \\u003cp\\u003eData were extracted from Spike2 as MATLAB files and analyzed using MATLAB (version 2023a, MathWorks). Signals were downsampled to 500 Hz and the DC offset was removed. The 50 Hz noise was reduced using a notch filter. Spectral analysis was performed using previously published scripts [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e] and the Chronux toolbox [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e] with the following parameters: time-bandwidth product\\u0026thinsp;=\\u0026thinsp;2, number of tapers\\u0026thinsp;=\\u0026thinsp;3, frequency band\\u0026thinsp;=\\u0026thinsp;0-150 Hz, moving window\\u0026thinsp;=\\u0026thinsp;4 s (0.1 s overlap). Signal power was transformed to decibels. The following bandwidths were used for later analyses: delta (0.9-4.0 Hz), theta (4.0\\u0026ndash;8.0 Hz), alpha (8.0\\u0026ndash;12 Hz), beta (12\\u0026ndash;30 Hz), and gamma (30\\u0026ndash;70 Hz). The percentage of relative power was normalized against the 20 min preceding the pilocarpine injection and analyzed in 20- or 40- min bins, as required. No differences were found between the baseline and post-scopolamine injection periods (data not shown), therefore, these data were combined for analysis.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eData analysis and figures\\u003c/h2\\u003e \\u003cp\\u003eParametric or non-parametric ANOVA was used as appropriate. Statistical analyses were performed using GraphPad Prism (version 9, GraphPad Software). Figures were generated in GraphPad Prism and MATLAB.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eEffect of CBD and THC on seizures induced by pilocarpine\\u003c/h2\\u003e \\u003cp\\u003eOur study aimed to investigate the effects of the cannabinoid drugs CBD and THC on epilepsy in mice. Specifically, we induced temporal lobe epilepsy in mice using the muscarinic agonist pilocarpine [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. We employed a protocol similar to the one described in our previous publication [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e], as illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. To mitigate the peripheral effects of muscarinic agonism, we administered scopolamine methylnitrate 1 mg/kg i.p. Thirty minutes later, pilocarpine 300 mg/kg i.p was injected. Subsequently, after 20 min, mice received one of three treatments: vehicle, CBD 100 mg/kg i.p, or THC 10 mg/kg i.p. We video-recorded mouse behaviors during this period and measured two parameters: the duration of seizures and the Racine scale score.\\u003c/p\\u003e \\u003cp\\u003eSeizure frequency was increased in the CBD group compared to vehicle and THC groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, One-way ANOVA: F\\u003csub\\u003e(2,19)=\\u003c/sub\\u003e8.924, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01; CBD vs vehicle or THC\\u0026thinsp;=\\u0026thinsp;p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 with Tukey's multiple comparisons test). However, we observed no changes between the vehicle and THC groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). Regarding seizure duration parameters, THC groups showed decrease in total seizure duration (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, One-way ANOVA: F\\u003csub\\u003e(2,19)=\\u003c/sub\\u003e9.612, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01; vehicle vs THC\\u0026thinsp;=\\u0026thinsp;p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 with Tukey's multiple comparisons test) and mean seizure duration (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, One-way ANOVA: F\\u003csub\\u003e(2,19)=\\u003c/sub\\u003e5.836, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; vehicle vs THC\\u0026thinsp;=\\u0026thinsp;p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 with Tukey's multiple comparisons test). Meanwhile, we found a decrease in the mean but not in total seizure duration for the CBD group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB-C, mean seizure duration: vehicle vs CBD\\u0026thinsp;=\\u0026thinsp;p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 with Tukey's multiple comparisons test). Although these data show the effect of CBD in pilocarpine-induced seizures, evaluation of seizure severity with the Racine scale showed no effect of CBD on seizure (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD). Meanwhile, a decrease in Racine scale value was found in THC group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD, Kruskal-Wallis test: p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01; vehicle and CBD vs THC\\u0026thinsp;=\\u0026thinsp;p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 with Dunn's multiple comparisons test), but it is relevant to note that animals exhibited marked sedation after THC injection. Overall, these behavioral data indicate that CBD decreases the duration but not the severity of seizures, while THC decreases seizure severity and duration, increasing mice drowsiness.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCBD and THC do not alter pilocarpine-induced cortical activity\\u003c/h2\\u003e \\u003cp\\u003eWe assessed the effects of CBD and THC on cortical activity using EEG in anesthetized mice. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA summarizes the experimental protocol. Under urethane anesthesia, a screw electrode was placed at the cortical level, near the dorsal hippocampus (for more details, see the Methods section). To minimize the peripheral effects of pilocarpine, mice were injected with scopolamine methylnitrate. Subsequently, pilocarpine 300 mg/kg i.p was administered, and 20 min later, either vehicle, CBD (100 mg/kg, i.p), or THC (10 mg/kg, i.p) was injected. Recordings were conducted for 2 h.\\u003c/p\\u003e \\u003cp\\u003eA representative example of an EEG recording 20 min before and after pilocarpine 300 mg/kg i.p. injection is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB, C. During baseline, the raw signal showed high-amplitude low frequency oscillations, which are characteristic of urethane anesthesia [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e] (\\u003cb\\u003eFig.\\u0026nbsp;3B1\\u003c/b\\u003e). Following pilocarpine injection, high frequency oscillations were more prominent (\\u003cb\\u003eFig.\\u0026nbsp;3B2\\u003c/b\\u003e). Spectral analysis confirmed these observations, as frequencies lower than 5 Hz showed high power values during baseline (\\u003cb\\u003eFig.\\u0026nbsp;3C1\\u003c/b\\u003e), whereas the power of frequencies higher than 10 Hz were increased after pilocarpine (\\u003cb\\u003eFig.\\u0026nbsp;3C2\\u003c/b\\u003e). To evaluate the effect of pilocarpine in specific bandwidths, we performed statistical analyses, considering all groups together (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD, N\\u0026thinsp;=\\u0026thinsp;13). We found a significant increase in the percentage of relative power was found at theta (4\\u0026ndash;8 Hz), beta (12\\u0026ndash;30 Hz) and gamma (30\\u0026ndash;70 Hz) frequencies (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001 with Wilcoxon sign rank test). We found comparable results when raw EEG power was analyzed or when this analysis was performed between groups (data not shown). Overall, these data show that a pilocarpine injection under anesthesia can induce epileptic-like activity in the EEG.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe studied the effect of CBD and THC on EEG activity after pilocarpine injection (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). The median EEG spectral power was obtained and color-plotted over time for vehicle (N\\u0026thinsp;=\\u0026thinsp;5), CBD 100 mg/kg i.p (N\\u0026thinsp;=\\u0026thinsp;4) and THC 10 mg/kg i.p (N\\u0026thinsp;=\\u0026thinsp;4) groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). Before and after treatment administration (at 40 min), we observed no differences between groups in the spectral density maps (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). Then, we calculated the percentage of relative power for all bandwidths, in the 40\\u0026ndash;80 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e\\u003cb\\u003eB1\\u003c/b\\u003e) and 80\\u0026ndash;120 (\\u003cb\\u003eFig.\\u0026nbsp;5B2\\u003c/b\\u003e) minute epochs. All frequency bands (except alpha) exhibited an increase in power compared to the baseline (pre-pilocarpine). The most significant increases were observed in the beta (up to 500%) and gamma (up to 1000%) bands. Between groups, neither CBD nor THC induced changes in EEG power bands compared to the vehicle, finding no statistically significant differences in any band for each bin tested (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB1-2, Kruskal-Wallis test: p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05).\\u003c/p\\u003e \\u003cp\\u003eThese findings indicate that while pilocarpine significantly elevates the power across all EEG frequency bands, particularly in the high-frequency beta and gamma ranges, neither CBD nor THC alters this heightened cortical activity. This shows that under the conditions of our study, CBD and THC do not modulate the acute electroencephalographic responses induced by pilocarpine.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTHC, but not CBD, reverses pilocarpine-induced changes in neuroLSD1 levels\\u003c/h2\\u003e \\u003cp\\u003eWe have previously demonstrated that pilocarpine leads to significant epigenetic modifications in the hippocampus, including alterations in the LSD1/CoREST/HDAC1/2 complex and histone methylation patterns [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. In this study, we further investigated changes in the neuroLSD1/LSD1 ratio. Seven hours after pilocarpine injection, mice were sacrificed, and the prefrontal cortex (PFC) and hippocampus were dissected to extract mRNA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, for more details in Methods).\\u003c/p\\u003e \\u003cp\\u003eWe measured the percentages of neuroLSD1 and LSD1 for each group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). A vehicle group was included for comparison. One-way ANOVA showed significant differences in both the hippocampus (F\\u003csub\\u003e(3, 12)\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;6.881, p\\u0026thinsp;=\\u0026thinsp;0.0060) and PFC (F\\u003csub\\u003e(3, 12)\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;5.787, p\\u0026thinsp;=\\u0026thinsp;0.0110). We observed a decrease in neuroLSD1 transcript after pilocarpine injection compared to control conditions in the hippocampus (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 Tukey's multiple comparisons test) but not in the PFC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB, p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05 Tukey's multiple comparisons test). These hippocampal results replicate our previous findings [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Regarding cannabinoid drugs, CBD did not significantly alter LSD1 levels compared to vehicle or pilocarpine alone in either region (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e, p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05, Tukey's multiple comparisons test). Notably, THC increased neuroLSD1 levels compared to both pilocarpine and pilocarpine\\u0026thinsp;+\\u0026thinsp;CBD in the hippocampus and PFC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Tukey's multiple comparisons test).\\u003c/p\\u003e \\u003cp\\u003eIn summary, while CBD did not affect LSD1 levels, THC significantly increased neuroLSD1 expression in both regions, suggesting a potential differential impact of cannabinoids on the epigenetic landscape following seizure induction.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"DISCUSSION\",\"content\":\"\\u003cp\\u003eThe present study reveals critical dissociation among behavioral, electrophysiological, and genomic effects of acute cannabinoid administration during a SE induced by pilocarpine. Our main findings indicate that neither CBD nor THC was sufficient to normalize cortical electroencephalographic activity during SE, although both drugs reduced the duration of seizures. Furthermore, THC inhibited the negative homeostatic regulation of neuroLSD1, suggesting the maintenance of cortical excitability, which may facilitate further seizures.\\u003c/p\\u003e \\u003cp\\u003eRegarding behavioral effects, the cannabinoids elicited distinct phenotypes. THC induced a marked sedation [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e] characteristic of CB1 receptor activation [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e], which effectively masked the behavioral expression of seizures. In contrast, CBD (100 mg/kg) failed to reduce seizure severity, diverging from previous reports demonstrating efficacy at lower doses [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. Instead, CBD produced a pattern of \\\"seizure fragmentation,\\\" characterized by a paradoxical increase in seizure frequency coupled with reduced individual seizure duration. This likely reflects the inverted U-shaped dose-response curve of cannabinoids [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. We propose that while CBD may activate termination mechanisms via targets like ENT1, the concurrent engagement of pro-excitatory TRPV1 channels at high concentrations [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e] lowers the seizure threshold, converting continuous status epilepticus into repetitive, fragmented events rather than achieving sustained seizure freedom.\\u003c/p\\u003e \\u003cp\\u003eFrom an electrophysiological standpoint, the resistance to treatment was significant (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Despite the behavioral suppression seen with THC, neither cannabinoid reversed the pathological increase in beta (12\\u0026ndash;30 Hz) and gamma (30\\u0026ndash;70 Hz) EEG power. This persistence of the sustained epileptiform activity aligns with the hypothesis of homeostatic downregulation of the endocannabinoid system during severe excitotoxicity. Intense neuronal firing triggers the rapid internalization and desensitization of presynaptic CB1 receptors, rendering the system refractory to acute agonist stimulation precisely at the seizure focus [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIt is important to acknowledge that these recordings were obtained under urethane anesthesia. Urethane potentiates GABA-A receptors and inhibits NMDA receptors, shifting the basal balance toward inhibition [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. Although this could theoretically lead to a \\\"floor effect\\\" [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e], our data indicate that pilocarpine caused substantial and sustained increases in high-frequency power that easily surpassed anesthetic suppression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). This phenomenon has been corroborated in other chemoconvulsant models, such as 4-aminopyridine, where high-frequency ictal discharges displace anesthetic slow-waves [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Furthermore, seminal work by Stringer \\u0026amp; Sowell, 1994 confirms that urethane anesthesia preserves the validity of electrographic seizure patterns compared to awake animals, reinforcing that the failure of cannabinoids to reduce EEG power here was due to the refractory nature of the seizure network rather than an artifact of the anesthetic regimen.\\u003c/p\\u003e \\u003cp\\u003eThe disparity between the sedative effects of THC (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e) and its failure to reduce EEG power (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e) highlights a significant risk in preclinical drug evaluation. This uncoupling has been described with synthetic cannabinoids, where motor seizures are suppressed while hippocampal paroxysms persist [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. Our results extend this to phytocannabinoids, serving as a critical methodological warning: behavioral quiescence induced by CB1 agonists should not be interpreted as seizure cessation without corroborating electrophysiological evidence.\\u003c/p\\u003e \\u003cp\\u003eDespite the lack of electrographic silence, a key molecular finding was that THC, but not CBD, prevented the seizure-induced downregulation of neuroLSD1 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). NeuroLSD1 is a dominant-negative splicing variant of the epigenetic eraser LSD1 that facilitates the transcription of neuroplasticity genes [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. The downregulation of neuroLSD1 observed in our vehicle group represents a maladaptive splicing switch that contributes to a global repressive state [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. However, this molecular rescue presents a biological paradox when interpreted in the context of genetic studies. NeuroLSD1-null mice exhibit a hypoexcitable phenotype and increased resistance to pilocarpine-induced seizures [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. This suggests that the drastic downregulation of neuroLSD1 we observed in the vehicle group may act as an adaptive homeostatic brake: an attempt by the network to limit excitability by reinstating the repressive LSD1 complex. Then, the effect of THC is double-edged. By restoring neuroLSD1 levels, THC effectively opposed this homeostatic downregulation. The specific ability of THC, but not CBD, to restore neuroLSD1 levels implies a mechanism whereby CB1 signaling exerts upstream control over the splicing machinery. While previous studies have shown that LSD1 regulates the endocannabinoid system [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e], our data suggests a bidirectional relationship, potentially mediated by the CB1-dependent activation of MAPK/ERK pathways [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e], which are known to regulate splicing factors. Conversely, the failure of CBD to rescue neuroLSD1 is consistent with its non-canonical pharmacological profile. Lacking intrinsic affinity for the CB1 receptor, CBD acts via a pleiotropic mechanism, involving targets such as GPR55, TRPV1, and ENT1 [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e], which likely bypasses the specific CB1-dependent MAPK/ERK signaling axis required to drive the splicing machinery. Ultimately, while the restoration of neuroLSD1 by THC may preserve the chromatin competence required for immediate early gene transcription (potentially mitigating the \\\"epigenetic scar\\\"), it may simultaneously maintain the permissive state for network hypersynchrony, offering a molecular explanation for the observed electro-clinical dissociation.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was funded by: ANID Fondecyt Postdoctorado 3160308 to V.N. and 3230573 to R.I.G, ANID Fondecyt Regular 1191497 to P.H.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting Interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eRafael Ignacio Gatica: conceptualization, methodology, writing \\u0026ndash; original draft, data acquisition, formal analysis, and investigation. Ver\\u0026oacute;nica Noches: conceptualization, methodology, writing-review \\u0026amp; editing, data acquisition, formal analysis, investigation, funding acquisition. Marcela Gonz\\u0026aacute;lez: investigation, project administration. Pablo Henny: funding acquisition, supervision, writing-review \\u0026amp; editing. Mar\\u0026iacute;a Estela Andr\\u0026eacute;s: conceptualization, methodology, supervision, writing-review \\u0026amp; editing, resources, funding acquisition.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\\u003cbr\\u003e\\u003cstrong\\u003eEthics approval\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll experimental procedures were approved by the Bioethical Committee of the Pontificia Universidad Cat\\u0026oacute;lica de Chile (protocol ID 03082015).\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eL\\u0026ouml;scher W, Potschka H, Sisodiya SM, Vezzani A (2020) Drug resistance in epilepsy: Clinical impact, potential mechanisms, and new innovative treatment options. 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Epileptic Disord 22:S10\\u0026ndash;S15. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1684/epd.2020.1135\\u003c/span\\u003e\\u003cspan address=\\\"10.1684/epd.2020.1135\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e \\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"epilepsy, pilocarpine, epigenetics, neuroLSD1, THC, CBD\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-9569465/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-9569465/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThere is growing interest in using cannabis and its active ingredients for epilepsy treatment. The main components of cannabis are cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), with some formulations of CBD approved for treating epilepsies refractory to traditional pharmacological treatments. However, it remains unclear whether the acute administration of THC or CBD can reverse a seizure once it has started. In this study, we used a temporal epilepsy model in mice induced by pilocarpine injection and analyzed the effects of CBD and THC during status epilepticus (SE) on behavior, cortical activity, and epigenetics. Behavioral analyses showed that CBD did not alter seizure severity, while THC only increased sedation. Regarding cortical electrical activity, neither CBD nor THC reverted the electroencephalographic changes induced by pilocarpine. Finally, we found that acute THC treatment prevented the seizure-induced homeostatic downregulation of neuroLSD1, maintaining it at physiological levels despite the insult. These results highlight that an acute dose of cannabinoid drugs is insufficient to reduce seizures and suggest that THC could promote the ongoing network excitability during seizures.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Behavioral, electrophysiological, and epigenetic characterization of acute cannabinoid treatment during pilocarpine-induced status epilepticus in mice\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-05-08 20:34:07\",\"doi\":\"10.21203/rs.3.rs-9569465/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"e438ddeb-64a7-464b-b3f6-b787c5a4a06d\",\"owner\":[],\"postedDate\":\"May 8th, 2026\",\"published\":true,\"recentEditorialEvents\":[{\"type\":\"decision\",\"content\":\"Rejected\",\"date\":\"2026-05-08T13:44:08+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-06T23:36:16+00:00\",\"index\":19,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"99654959073932701513331349283190851312\",\"date\":\"2026-05-04T10:39:55+00:00\",\"index\":16,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"173427447388015846278698429570826783204\",\"date\":\"2026-05-01T12:09:43+00:00\",\"index\":14,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"10\",\"date\":\"2026-04-30T21:50:47+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-04-30T16:07:10+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-04-30T05:26:52+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Neurochemical Research\",\"date\":\"2026-04-29T19:34:25+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-05-12T10:00:47+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-05-08 20:34:07\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-9569465\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-9569465\",\"identity\":\"rs-9569465\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}